The Rosetteless gene controls development in the choanoflagellate S. rosetta.

A forward genetic screen identified the Rosetteless mutant, which fails to form rosettes but grows normally and forms normal chain colonies.

"Rosetteless cultures did not form rosettes in ACM, but otherwise appeared in wild type, forming normal chain colonies"
The causative lesion is a splice-donor mutation in rtls (EGD82922), which encodes a secreted protein with two C-type lectin-like domains.

"The rtls gene encodes a 119 kDa protein with an N-terminal signal peptide and two C-type lectin-like domains (CTLDs; Figure 5A)."
Function-blocking antibodies against Rtls inhibit rosette development in wild type cells without affecting growth.

"Therefore, we conclude that the function of secreted Rtls is specific to and essential for rosette development."
In rosettes, Rtls forms a thick extracellular layer at the basal poles of the cells.

"In rosettes, Rtls (cyan) was detected as a thick layer associated with the basal poles of the cells."

Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta.

An independent CRISPR-engineered premature-termination allele of rosetteless abolishes rosette development and secretion of Rosetteless protein.

"Mutations in rosetteless prevent the secretion of Rosetteless protein at the basal end of cells and into the interior or rosettes."

Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta.

In jumble and couscous glycosyltransferase mutants, Rosetteless reaches the basal pole but is not upregulated or secreted upon rosette induction.

"In both Jumble and Couscous cells, Rosetteless protein properly localized to the basal pole, but its expression did not increase nor was it secreted upon treatment with RIFs, as normally occurs in wild type cells"

PMID:41037400

A selection-based knockout approach for a choanoflagellate reveals regulation of multicellular development by Hippo signaling.

rosetteless was inactivated again with a selection-based CRISPR knockout pipeline, as one of three known regulators of multicellular development.

"We inactivated three known S. rosetta multicellular developmental regulators (rosetteless, couscous, and jumble)"

PMID:31896587

Biophysical principles of choanoflagellate self-organization.

All three genes known to be required for rosette development act on the extracellular matrix.

"Interestingly, all 3 genes known to be required for rosette development are regulators of the extracellular matrix (ECM)"